Multiple fluoroimmunoassay method capable of quickly differentiating infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), myeoplasma gallisepticum (MG) and mycoplasma synoviae (MS) and reagent

A technology of multiple fluorescence and immunoassays, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as higher requirements and increased difficulty of experiments, and achieve lower detection costs and higher sample usage. Small quantity, avoiding the effect of cross-hybridization

Active Publication Date: 2016-10-12
GUANGDONG LAB ANIMALS MONITORING INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in terms of sensitivity, specificity, and speed. However, in practical applications, when the sample volume is very large, single-plex fluorescent PCR has certain disadvantages in terms of cost and time. A multiplex fluorescent PCR method is much more complicated than

Method used

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  • Multiple fluoroimmunoassay method capable of quickly differentiating infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), myeoplasma gallisepticum (MG) and mycoplasma synoviae (MS) and reagent
  • Multiple fluoroimmunoassay method capable of quickly differentiating infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), myeoplasma gallisepticum (MG) and mycoplasma synoviae (MS) and reagent
  • Multiple fluoroimmunoassay method capable of quickly differentiating infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), myeoplasma gallisepticum (MG) and mycoplasma synoviae (MS) and reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Primers

[0096] After screening a large number of designed primers, it was found that the primer pairs L1 and L2, B1 and B2, G1 and G2, S1 and S2 were suitable for the rapid detection of chicken infectious laryngotracheitis virus (ILTV) and chicken infectious bronchitis virus simultaneously. (IBV), Mycoplasma gallisepticum (MG) and Mycoplasma gallisepticum (MS), the base sequences of which are shown below.

[0097] Primer L1: ACTGTGTAAAGCAGGCCAAGGTCTGT (SEQ ID NO: 1);

[0098] Primer L2: AACAATCAATTTGGCACAATGCC (SEQ ID NO: 2);

[0099] Primer S1: CCCAGCCATATACTCAGTCGTGC (SEQ ID NO: 3);

[0100] Primer S2: TCCACAACTTTTGTGACAGGACAC (SEQ ID NO: 4);

[0101] Primer P1: AGATCACAGAGCCCGTCAAAAT (SEQ ID NO: 5);

[0102] Primer P2: GCATATAACATCCAATACGAGTTTGAA (SEQ ID NO: 6);

[0103] Primer R1: ACGTCTCAAATACACTCTGATAC (SEQ ID NO: 7);

[0104] Primer R2: TTCCAATCKGGAGATTGCAAGTTG (SEQ ID NO: 8).

[0105] The present invention adopts the method of multiple fluores...

Embodiment 2

[0111] Example 2 Multiplex fluorescent immunoassay reagents for rapidly distinguishing chicken infectious laryngotracheitis virus (ILTV), chicken infectious bronchitis virus (IBV), mycoplasma gallisepticum (MG) and mycoplasma gallisepticum (MS)

[0112] This reagent includes the following components:

[0113] (1) The primers designed in Example 1 for multiplex fluorescent immunoassay;

[0114] (2) 4 kinds of fluorescently encoded microspheres containing anti-tag sequences with different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in the multiple fluorescent immunoassay primers; all 4 kinds of microspheres can be purchased From luminex company, the numbers of fluorescently encoded microspheres corresponding to ILTV, IBV, MS and MG are MTAG-A067, MTAG-A036, MTAG-A025 and MTAG-042, respectively.

[0115](3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0116] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for ILTV, IBV, MS and MG

[0117] (1) Construction of ILTV, IBV, MS and MG plasmids

[0118] The RNA / DNA of ILTV, IBV, MG, and MS pathogens were extracted with Tiangen’s automatic nucleic acid extractor, and the primer pairs L1 and L2, B1 and B2, G1 and G2, S1 and S2 were used for RT-PCR amplification, respectively. The amplified products were detected by agarose gel electrophoresis and purified by gel cutting. The purified cDNAs were respectively connected to the pMD-19T vector, and the connected products were transformed into DH5a competent cells. Single clones were selected for colony PCR identification. Colonies identified as positive bacteria were subjected to plasmid extraction and sent for sequencing.

[0119] (2) Plasmid PCR amplification

[0120] The primers described in Example 1 were used to perform single-fold and quadruple RT-PCR amplification on ILTV, IBV, MS, and MG primers, r...

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Abstract

The invention discloses a multiple fluoroimmunoassay method capable of quickly differentiating infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), myeoplasma gallisepticum (MG) and mycoplasma synoviae (MS) and a reagent. The method is simple to operate, a target amplified fragment is obtained through polymerase chain reaction (PCR); an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized, a mean fluorescence intensity (MFI) value is read through a detector, and pathogens of different types are differentiated. According to the method, avian infectious laryngotracheitis virus, infectious bronchitis virus, myeoplasma gallisepticum and mycoplasma synoviae can be accurately detected simultaneously, and the method is high in specificity and sensitivity and good in repeatability. Compared with a conventional detection method, the method can be used to simultaneously detect various target molecules in an identical sample, the sample usage amount is small, the method is simple and quick to operate, and the detection cost can be greatly reduced. The method is good in flexibility, and the varieties of detected pathogens can be increased or decreased as required on the basis.

Description

technical field [0001] The invention belongs to the field of pathogen detection in aquaculture, in particular to a method for rapidly distinguishing chicken infectious laryngotracheitis virus (ILTV), chicken infectious bronchitis virus (IBV), mycoplasma gallisepticum (MG) and mycoplasma gallisepticum ( MS) multiple fluorescence immunoassay method and reagents. Background technique [0002] As the market demand for poultry continues to increase, the poultry industry has developed rapidly, the degree of intensification of the poultry industry has continued to increase, and the infection of poultry respiratory pathogens has been on the rise year by year. Respiratory diseases of poultry have been a major factor causing significant economic losses in the poultry industry. The pathogenesis of chicken respiratory disease has its complexity. It can be caused by viruses, mycoplasma and bacteria, and various mixed infections will appear clinically. The occurrence of these diseases c...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6804C12Q1/689C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 郭鹏举朱余军丛峰黄韧陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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